Method for laminaribiose synthesis

ABSTRACT

The invention concerns a method for preparing Laminaribiose comprising a step for glycoside binding between a donor and an acceptor of glycosyl. The invention is characterized in that the glycosyl donor is in pyranose form and corresponds to formula (II); the glycosyl acceptor is in furanose form and corresponds to formula (III); said binding step is performed in solution in an anhydrous organic solvent, at a temperature ranging between −80° C. and 40° C., for a time interval ranging between 1 minute and 8 hours, in the presence of a suitable promoter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a §371 application of PCT/FR99/00857 filed on Apr.13, 1999, which claims priority to French application 98/04610 filed onApr. 14, 1998.

FIELD OF THE INVENTION

The present invention relates to a novel method of chemical preparationof β-D-glucopyranosyl-(1→3)-D-glucopyranose of formula (I), commonlycalled laminaribiose.

BACKGROUND OF THE INVENTION

Laminaribiose is a disaccharide which is used notably in theagricultural field and as an antiseptic.

This disaccharide is in general obtained by hydrolysis or by acetolysisof natural polysaccharides of plant origin (see Villa, Phaff, Notario,Carbohydr. Res., 1979, 74, 369; Kusama, Kusakabe, Zama, Murakami, Yasui,Agric. Biol. Chem., 1984, 48, 1433; Wang, Sakairi, Kuzuhara, Carbohydr.Res., 1991, 219, 133; Moreau, Viladot, Samain, Planas, Driguez, Bioorg.Med. Chem., 1996, 4, 1849).

Laminaribiose can also be prepared chemically, notably by methodsderived from the Koenigs-Knorr method of O-glycosylation (see Koenigs,Knorr, Ber. Dtsch. Chem. Ges., 1901, 34, 957) which makes use ofglycosyl halides as glycosyl donors.

A first method has thus been proposed by Freudenberg and von Oertzen in1951 (see Freudenberg, von Oertzen, Justus Liebigs Ann. Chem., 1951,574, 37), and a second method has been described by Bächli and Percivalin 1952 (see Bäichli, Percival, J. Chem. Soc., 1952, 1243).

The major drawbacks of these two methods reside in a purification whichis difficult to carry out and in an overall yield which is lower that10%.

A third method has been proposed by Takeo in 1979 (see Takeo, Carbohydr.Res., 1979, 77, 245), but it necessitates several steps of selectiveprotection and deprotection of the hydroxyls of the acceptor used whichis in glucopyranose form.

It has also been proposed to form laminaribiose from ortho-esters (seeKochetkov, Bochtov, Sokolovskaya, Snyatkova, Carbohydr. Res., 1971, 16,17). This method does however prove to be difficult to carry out andenables laminaribiose to be obtained only with an overall yieldneighbouring 10%.

Under these circumstances, the aim of the present invention is toprovide a novel method of chemical preparation of laminaribiose whichhas a limited number of steps which are easy to carry out and whichenables the product sought after to be obtained in pure form with a highoverall yield.

SUMMARY OF THE INVENTION

The solution in accordance with the present invention to solve thistechnical problem consists of a method of preparing laminaribiosecomprising a step of glycosidic coupling between a glycosyl donor and aglycosyl acceptor, characterised in that:

the glycosyl donor is in pyranose form and is of formula (II):

 in which:

R₁ represents:

an alkyl or haloalkyl radical having 1 to 6 carbon atoms

an aryl radical which is non-substituted or substituted with one or moregroups selected from a halogen atom, an alkoxy radical having 1 to 6carbon atoms or a nitro group;

X represents an electrophilic leaving group selected from:

a group of formula S(O)_(n)R′ in which R′ represents an alkyl radicalhaving 1 to 6 carbon atoms, an aryl radical which is non-substituted orsubstituted with an alkoxy group having 1 to 6 carbon atoms, a nitro oracetamide group, and n is an integer equal to 0 or 1; or

a trichloroacetimidate group;

the glycosyl acceptor is in furanose form and is of formula (III)

 in which:

R₂ and R₃ together form a methylidyl, ethylidyl, trichloroethylidyl,isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical ; and

R₄ and R₅ together form a methylidyl, ethylidyl, trichloroethylidyl,isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical, orindependently represent a benzyl, acetyl, benzoyl, chlorobenzoyl,methoxybenzoyl, nitrobenzoyl, allyl, chlorobenzyl, methoxybenzyl ornitrobenzyl radical;

said coupling step is carried out in solution in an anhydrous organicsolvent, at a temperature between −80° C. and 40° C., for a period of 1minute to 8 hours, in the presence of a suitable promoter selected from:

a source of halonium ions, combined or not with a Lewis acid or a saltof a strong acid, in the case in which X represents an S(O)_(n)R′ groupas defined above in which n is equal to 0;

a Lewis acid combined with an amine, in the case in which X representsan S(O)_(n)R′ group as defined above in which n is equal to 1; or

a Bronsted acid or a Lewis acid, in the case in which X represents atrichloroacetimidate group ; and

the reaction product thus obtained, neutralised and purified, beingsubjected to a deprotection treatment to give, after purification,laminaribiose.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered, and this constitutes the basis of the presentinvention, that it was possible to chemically prepare laminaribiose witha limited number of steps which enable a relatively high overall yieldto be obtained, by a judicial choice of the glycosyl donor and of theglycosyl acceptor, as well as of the promoter used during the couplingreaction.

In the description and claims:

<<alkyl radical having 1 to 6 carbon atoms>> is understood as meaningany linear or branched hydrocarbon chain, such as a methyl, ethyl,propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl,hexyl, or isohexyl radical, for example;

<<haloalkyl radical having 1 to 6 carbon atoms>> is understood asmeaning any alkyl radical 1 to 7 hydrogen atoms of which are substitutedby 1 to 7 halogen atoms, such as a chloromethyl radical, a bromomethylradical, a trifluoromethyl radical, a 2,2,2-trifluoroethyl radical, apentafluoroethyl radical, or a heptafluoropropyl radical, for example;

<<aryl radical>> is understood as meaning an aromatic ring having 5 or 6carbon atoms or heteroatoms, such as a phenyl, pyridyl, thienyl,furanyl, or pyrimidyl radical, for example.

The glycosyl donor of formula (II) mentioned above as well as theglycosyl acceptor of formula (III) mentioned above can be obtainedrelatively easily, in one or two steps, from D-glucose.

Advantageously, the glycosyl donor will in general be selected from thecompounds of formula (II) mentioned above in which:

R₁ represents a radical selected from the group consisting of methyl,chloromethyl, trifluoromethyl, tert-butyl, phenyl, chlorophenyl,methoxyphenyl and nitrophenyl radicals;

X represents a radical selected from the group consisting of thiomethyl,thioethyl, thiopropyl, thiophenyl, thionitrophenyl, and thiopyridylradicals.

In general, the promoter used during the coupling step mentioned abovewill be selected from:

N-bromosuccinimide or N-iodosuccinimide, combined or not with a Lewisacid selected from ferric chloride, copper ditriflate, tin ditriflate,boron trifluoride dietherate, tin or zirconium tetrachloride, methyltriflate, trimethyl- (or triethyl-) silyl triflate, silver triflate,cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zincditriflate, bismuth tritriflate, iron tritriflate, gallium tritriflate,or with a salt of a strong acid such as tetrabutylammonium triflate, inthe case in which X represents an S(O)_(n)R′ group as defined above inwhich n is equal to O,

a Lewis acid selected from triflic anhydride, ferric chloride, copperditriflate, tin ditriflate, boron trifluoride dietherate, tin orzirconium tetrachloride, methyl triflate, trimethyl- (or triethyl-)silyl triflate, silver triflate, cadmium ditriflate, cobalt ditriflate,nickel ditriflate, zinc ditriflate, bismuth tritriflate, irontritriflate, gallium tritriflate, combined with an amine particularlysuch as di-tert-butylmethylpyridine, in the case in which X representsan S(O)_(n)R′ group as defined above in which n is equal to 1, and

a Bronsted acid particularly such as triflic acid orpara-toluenesulphonic acid or a Lewis acid selected from triflicanhydride, ferric chloride, copper ditriflate, tin ditriflate, borontrifluoride dietherate, tin or zirconium tetrachloride, methyl triflate,trimethyl- (or triethyl-) silyl triflate, silver triflate, cadmiumditriflate, cobalt ditriflate, nickel ditriflate, zinc ditriflate,bismuth tritriflate, iron tritriflate, gallium tritriflate, in the casein which X represents a trichloroacetimidate group.

In a currently preferred embodiment of the method according to theinvention:

the glycosyl donor is of formula (II) mentioned above in which:

R₁ represents a phenyl radical; and

X represents an S(O)_(n)R′ radical in which n is equal to O and R′represents an ethyl or phenyl radical;

the glycosyl acceptor is of formula (III) mentioned above in which:

R₂, R₃ and R₄, R₅ together form a cyclohexylidyl or isopropylidylradical.

In this particular embodiment, the promoter used during the couplingreaction is constituted of a mixture of N-iodosuccinimide and of tinditriflate, preferably in proportions between 1:0.5 and 1:0.005.

In general, the coupling step mentioned above is carried out in solutionin dichloromethane, 1,2-dichloroethane or toluene, preferably in thepresence of molecular sieves, at a temperature between −30° C. and 30°C., for a period of 1 minute to 6 hours, preferably at 10° C. for 30minutes.

It will be possible for the respective amounts of glycosyl donor, ofglycosyl acceptor and of promoter to be determined easily by the personskilled in the art.

In general, the coupling reaction can be carried out by allowing toreact:

one equivalent of glycosyl acceptor;

one to two equivalents of glycosyl donor; and

one to two equivalents of promoter;

in 5 to 200 equivalents by weight, with respect to the acceptor, of asolvent.

Advantageously, an organic solvent such as dichloromethane,1,2-dichloroethane or toluene will be used, in the presence of molecularsieves (intended for trapping the acid which can form during thereaction) such as 4 Å molecular sieves for example, used in an amount of10 to 200 mg/ml of solvent.

The product obtained by the coupling reaction mentioned above isgenerally neutralised and then purified.

The neutralisation can be carried out by adding an organic base,preferably triethylamine or ethanolamine, or even by adding an inorganicbase, preferably sodium or potassium carbonate or hydrogen carbonate,followed by filtering the salt obtained.

The purification can be carried out:

either by chromatography, for example on a silica gel or active charcoalcolumn,

or by fractional crystallisation preferably in an organic solvent or amixture of organic solvents such as ethyl ether, ethyl acetate,cyclohexane or ethanol.

The product of the coupling reaction, neutralised and purified leads,via a deprotection treatment followed by a purification, tolaminaribiose.

In general, the deprotection treatment mentioned above comprises twosteps, the first consisting of a partial deprotection of the product ofthe coupling reaction, by cleavage of the acetal groups originating fromthe glycosyl acceptor.

Within the context of the method in accordance with the presentinvention, the deprotection treatment comprises:

a) cleaving the acetal groups originating from the glycosyl acceptor byan acidic treatment in an aqueous or hydro-organic medium, or in thepresence of an acidic resin;

b) purifying the product thus obtained;

c) transesterifying or hydrolysing the product obtained in step b); and

d) purifying the product thus obtained.

The cleavage reaction a) mentioned above will preferably be carried outin an acidic hydro-organic medium, such as in a mixture of equal volumesof trifluoroacetic acid and water for example, at a temperature between10 and 70° C. for a period of 1 hour to 10 days and in this case, thepartially deprotected product obtained will be purified by fractionalcrystallisation, preferably in methanol, or by chromatography.

It is also possible for acetic acid, oxalic acid, formic acid, sulphuricacid, hydrochloric acid, and phosphoric acid, to be used instead oftrifluoroacetic acid.

Within the context of the method in accordance with the presentinvention, the transesterification step c) mentioned above will becarried out in an alcoholic solvent such as methanol or ethanol in thepresence of a catalytic amount of sodium or of sodium or potassiummethoxide or ethoxide, for a period of 1 minute to 10 days.

The product of transesterification thus obtained will generally bepurified by a method comprising:

d1) neutralising the product obtained in step c);

d2) removing the benzoic ester formed, either by azeotropic evaporationwith water, or by extraction with an organic solvent;

d3) concentrating under reduced pressure the residual aqueous phase;

d4) optionally, lyophilising or crystallising the laminaribiose thusobtained in a hydro-alcoholic mixture.

Other characteristics and advantages of the invention will be betterunderstood upon reading the following non-limiting Examples.

EXAMPLE 1

Example of Preparation of a Glycosyl Acceptor

In this example, the glycosyl acceptor, namely1,2:5,6-di-O-cyclohexylidene-α-D-glucofuranose (compound of formula IIIin which R₂, R₃ and R₄, R₅ represent a cyclohexylidyl radical) wasprepared in one single step from D-glucose.

20 ml (375 mmol; 1.03 eq) of concentrated sulphuric acid are addeddropwise to 65 g of D-glucose (361 mmol; 1 eq) and 85 ml (820 mmol ;2.27 eq) of cyclohexanone in 50 ml of 1,4-dioxane (587 mmol; 1.62 eq) atambient temperature. Once the addition is complete, after 30 minutes,the product precipitates from the reaction medium. The precipitate isbroken up, filtered off and washed with water. The crude product is thenpurified by recrystallisation from cyclohexane to give to 104 grams of1,2: 5,6-di-O-cyclohexylidene-α-D-glucofuranose.

Yield (%): 85

White solid.

M. Pt. (°C.)=134-136

TLC : R_(f)=0.8 (dichloromethane/methanol (9/1; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): 112.49, 110.32 (C quat.); 104.93 (C1);84.62 (C2); 81.23 (C4); 75.39 (C3); 73.27 (C5); 67.40 (C6); 36.50,36.48, 35.69, 34.65, 25.09, 24.94, 24.08, 23.95, 23.82, 23.63, (CH₂).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 5.93, (d, 1H, H1, J_(H1-H2)=3.6 Hz);4.50 (d, 1H, H2, J_(H2-H1)=3.6 Hz); 4.34-4.30 (m,2H, H3, H5); 4.14 (dd,1H, H6, J_(H6-H5)=6.2 Hz, J_(H6-H6′)=8.6 Hz); 4.04 (dd, 1H, H4,J_(H4-H3)=2.8 Hz, J_(H4-H5)=7.6 Hz); 3.95 (dd, 1H, H6′, J_(H6′-H5)=5.4Hz, J_(H6′-H6)=8.6 Hz); 1.70-1.36 (m, 20H, CH₂).

EXAMPLE 2

Example of Preparation of a Glycosyl Donor

In this example, the glycosyl donor (compound of formula II in which R₁is a phenyl and X is an S(O)_(n)R′ group in which n=0 and R′ representsan ethyl group) was prepared in two steps.

a) Preparation of 1,2,3,4,6-penta-O-benzoyl-D-glucopyranose

100 g of D-glucose (555 mmol; 1 eq) are dissolved in 2 l of pyridine andthe reaction medium is heated under reflux for 1 hour before 387 ml(3,330 mmol; 6 eq) of benzoyl chloride are added thereto in the hot.After addition, the medium is diluted with water, the productprecipitates and is filtered off and rinsed with water to neutrality.After drying, it is purified by recrystallisation from ethyl acetate.

Yield (%): 100

White solid.

M. Pt. (°C.)=164-166 (β anomer); 188-191 (α anomer).

TLC: R_(f)=0.3 (petroleum ether/ethyl acetate (8/2; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): β anomer : 166.17, 165.74, 165.19,165.16, 164.66 (C═O); 92.74 (C1); 73.21, 72.85 (C3, C5); 70.87 (C2);69.08 (C4); 62.71 (C6); α anomer : 166.15, 165.96, 165.42, 165.18,164.47 (C═O); 90.08 (C1); 70.54, 70.51, 70.45 (C2, C3, C5); 68.83 (C4);62.49 (C6).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 8.20-7.16 (m, 25H, H arom.); β anomer:6.30 (d, 1H, H1, J_(H1-H2)=8.0 Hz); 6.05 (t, 1H, H3,J_(H3-H2)=J_(H3-H4)=9.5 Hz); 5.87 (dd, 1H, H2, J_(H2-H1)=8.0 Hz,J_(H2-H3)=9.5 Hz); 5.84 (t, 1H, H4, J_(H4-H3)=J_(H4-H5)=9.6 Hz); 4.66(dd, 1H, H6, J_(H6-H5)=2.9 Hz, J_(H6-H6′)=12.3 Hz); 4.52 (dd, 1H, H6′,J_(H6′-H5)=4.7 Hz, J_(H6′-H6)=12.3 Hz); 4.41 (ddd, 1H, H5, J_(H5-H4)=9.7Hz, J_(H5-H6)=3.0 Hz, J_(H5-H6′)=4.6 Hz); α anomer: 6.85 (d, 1H, H1,J_(H1-H2)=3.8 Hz); 6.33 (t, 1H, H3, J_(H3-H2)=J_(H3-H4)=10.0 Hz); 5.88(t, 1H, H4, J_(H4-H3)=J_(H4-H5)=9.8 Hz); 5.69 (dd, 1H, H2, J_(H2-H1)=3.8Hz, J_(H2-H3)=10.3 Hz); 4.65-4.60 (m, 2H, H5, H6); 4.48 (dd, 1H, H6′,J_(H6′-H5)=5.0 Hz, J_(H6′-H6)=13.0 Hz).

b) Preparation of ethyl 2,3,4,6-tetra-O-benzoyl-1-thio-D-glucopyranoside

100 g of perbenzoyl glucose (143 mmol; 1 eq) are dissolved in 2 l ofdichloromethane and the reaction medium is brought to reflux and 12.7 mlof ethanethiol (171 mmol; 1.2 eq) and 54.2 ml (429 mmol; 3 eq) of borontrifluoride etherate are added thereto. After 2 hours of reaction intotal, the medium is diluted with dichloromethane, washed with a 5%solution of sodium hydrogen carbonate and then with water until pHneutral. The product is recrystallised from ethyl acetate and 73 gramsof product are thus collected.

Yield (%): 80

White solid.

M. Pt. (°C.)=133-134 (α anomer)

TLC: R_(f)=0.4 (β anomer); 0.5 (α anomer)(petroleum ether/ethyl acetate(8/2; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): 166.23, 166.18, 165.87, 165.71, 165.50,165.38, 165.27 (C═O); α anomer: 82.10 (C1); 71.74 (C2); 70.99 (C3);69.62 (C4); 68.21 (C5); 63.14 (C6); 24.34 (CH₂); 14.71 (CH₃); β anomer:84.02 (C1); 76.37 (C5); 74.19 (C3); 70.68 (C2); 69.71 (C4); 63.42 (C6);24.47 (CH₂); 15.01 (CH₃).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 8.06-7.26 (2m, 40H, H arom.); α anomer:6.08 (t, 1H, H3, J_(H3-H2)=J_(H3-H4)=9.9 Hz); 5.95 (d, 1H, H1,J_(H1-H2)=5.8 Hz); 5.68 (t, 1H, H4, J_(H4-H3)=J_(H4-H5)=9.9 Hz); 5.51(dd, 1H, H2, J_(H2-H1)=5.8 Hz, J_(H2-H3)=10.2 Hz); 4.88 (ddd, 1H, H5,J_(H5-H4)=10.2 Hz, J_(H5-H6)=2.8 Hz, J_(H5-H6′)=5.4 Hz); 4.60 (dd, 1H,H6, J_(H6-H5)=2.8 Hz, J_(H6-H6′)=12.2 Hz); 4.52 (dd, 1H, H6′,J_(H6-H5)=5.4 Hz, J_(H6′-H) ₆=12.2 Hz); 2.62 (qd, 2H, CH₂, J=7.4 Hz,J=9.6 Hz); 1.25 (t, 3H, CH₃, J=7.4 Hz); β anomer: 5.93 (t, 1H, H3,J_(H3-H2)=J_(H3-H4)=9.7 Hz); 5.68 (t, 1H, H4, J_(H4-H3)=J_(H4-H5)=9.8Hz); 5.57 (t, 1H, H2, J_(H2-H1)=J_(H2-H3)=9.7 Hz); 4.87 (d, 1H, H1,J_(H1-H2)=10 Hz); 4.64 (dd, 1H, H6, J_(H6-H5)=3.1 Hz, J_(H6-H6′)=12.1Hz); 4.50 (dd, 1H, H6′, J_(H6′-H5)=5.5 Hz, J_(H6′-H6)=12.1 Hz); 4.18(ddd 1H, H5, J_(H5-H4)=10.0 Hz, J_(H5-H6)=3.0 Hz, J_(H5-H6′)=5.5 Hz);2.77 (qd, 2H, CH₂, J=7.4 Hz, J=9.6 Hz); 1.26 (t, 3H, CH₃, J=7.4 Hz).

EXAMPLE 3

Example of a Coupling Reaction According to the Invention

A coupling reaction between the donor of Example 2 and the acceptor ofExample 1 was carried out to give the following novel compound:

2,3,4,6-tetra-O-benzoyI-β-D-glucopyranosyl-(1→3)-1.2:5,6-di-O-cyclohexylidene-α-D-glucofuranose

64 g (100 mmol; 1.06 eq) of ethyl 2,3,4,6-tetra-Obenzoyl-1-thio-D-glucopyranoside, 32 g (94 mmol; 1 eq) of1,2:5,6-di-O-cyclohexylidene-α-D-glucofuranose, 22.5 g (100 mmol; 1.06eq) of N-iodosuccinimide and 400 g of 4 Å molecular sieves areintroduced into a round-bottomed flask in the dark and then dissolved in400 ml of anhydrous dichloromethane at a temperature of 10° C., and 3.92grams (9.4 mmol; 0.1 eq) of tin ditriflate are then added. After 1 hourof reaction, the medium is neutralised with triethylamine, filtered andconcentrated. Purification on a silica gel column (flash, eluent:toluene/ethyl acetate (95/5 then 9/1: v/v)) enables 60.5 grams ofproduct to be collected.

Yield (%): 70

White solid.

TLC: R_(f)=0.4 (toluene/ethyl acetate (9/1; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): 166.19, 165.86, 165.20, 164.83 (C═O);112.71, 109.32 (C quat.); 104.66 (C1a); 100.08 (C1b); 82.83 (C2a); 81.68(C3a); 80.66 (C4a); 72.79 (C3b); 72.63 (C5b); 72.46 (C5a); 71.99 (C2b);69.57 (C4b); 66.26 (C6a); 63.12 (C6b); 36.43, 36.33, 35.49, 34.61,25.23, 24.84, 24.17, 23.89, 23.86, 23.54 (CH₂).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 8.03-7.26 (2m, 20H, H arom.); 5.90 (t,1H, H3b, J_(H3b-H2b)=J_(H3b-H4b)=9.6 Hz); 5.71 (t, 1H, H4b,J_(H4b-H3b)=J_(H4b-H5b)=9.6 Hz); 5.52 (dd, 1H, H2b, JH2b-H1b=8.2 Hz,J_(H2b-H3b)=9.4 Hz); 5.45 (d, 1H, H1a, J_(H1a-H2a)=3.6 Hz); 4.99 (d, 1H,H1b, J_(H1b-H2b)=7.8 Hz); 4.64 (dd, 1H, H6b, J_(H6b-H5b)=2.4 Hz,J_(H6-H6′b)=12.2 Hz); 4.49 (dd, 1H, H6′b, J_(H6′b-H5b)=5.0 Hz,J_(H6b-H6b)=12.2 Hz); 4.38 (q, 1H, H5a,J_(H5a-H4a)=J_(H5a-H6a)=J_(H5a-H6′a)=6.1 Hz); 4.35 (d, 1H, H3a,J_(H3a-H4a)=3.0 Hz); 4.32 (d, 1H, H2a, J_(H2a-H1a)=3.6 Hz); 4.18 (dd,1H, H4a, J_(H4a-H3a)=2.9 Hz, J_(H4a-H5a)=6.3 Hz); 4.11 (ddd, 1H, H5b,J_(H5b-H4b)=Hz, J_(H5b-H6b)=Hz, J_(H5b-H6′b)=Hz); 4,03 (dd, 1H, H6a,J_(H6a-H5a)=6.8 Hz, J_(H6a-H6′a)=8.3 Hz); 3.94 (dd, 1H, H6′a,J_(H6′a-H5a)=5.6 Hz, J_(H6′a-H6a)=8.3 Hz); 1.62-1.30 (m, 20H, CH₂).

EXAMPLE 4

Further Example of a Coupling Reaction According to the Invention

In this example, 2,3,4,6-tetra-O-benzoyl-β-Dglucopyranosyl-(1→3)-1,2:5,6-di-O-isopropylidene-α-D-glucofuranose:

was prepared by a coupling reaction.

In this example, the glycosyl acceptor compound is diacetone D-glucosewhich can easily be obtained from D-glucose and acetone.

64 g (100 mmol; 1.06 eq) of ethyl2,3,4,6-tetra-O-benzoyl-1-thio-D-glucopyranoside prepared in Example 2,24.5 g (94 mmol; 1 eq) of diacetone-D-glucose, 22.5 g (100 mmol; 1.06eq) of N-iodosuccinimide and 400 g of 4 Å molecular sieves areintroduced into a round-bottomed flask in the dark and then dissolved in400 ml of anhydrous dichloromethane. The reaction medium is cooled to 0°C. and 3.92 g (9.4 mmol; 0.1 eq) of tin ditriflate are added thereto.After 20 minutes of reaction, the medium is neutralised withtriethylamine, filtered and concentrated. Purification on a silica gelcolumn (flash, eluent:dichloromethane/ethyl acetate (95/5: v/v)) enables28 grams of product to be collected.

Yield (%): 35

White solid.

M. Pt. (°C.)=90-94

TLC: R_(f)=0.5 (toluene/ethyl acetate (8/2; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): 166.16, 165.85, 165.19, 164.81 (C═O);112.00, 108.72 (C quat.); 104.99 (C1a); 99.99 (C1b); 82.78 (C2a); 81.48(C3a); 80.47 (C4a); 73.05 (C5a); 72.72 (C3b); 72.65 (C5b); 71.85 (C2b);69.54 (C4b); 66.25 (C6a); 62.98 (C6b); 26.73, 26.67, 26.03, 25.15 (CH₃).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 8.03-7.26 (2m, 20H, H arom.); 5.90 (t,1H, H3b, J_(H3b-H2b)=J_(H3b-H4b)=9.7 Hz); 5.71 (t, 1H, H4b,J_(H4b-H3b)=J_(H4b-H5b)=9.7 Hz); 5.50 (dd, 1H, H2b, J_(H2b-H1b)=7.8 Hz,J_(H2b-H3b)=9.7 Hz); 5.48 (d, 1H, H1a, J_(H1a-H2a)=3.7 Hz); 4.98 (d, 1H,H1b, J_(H1b-H2b)=7.8 Hz); 4.66 (dd, 1H, H6b, J_(H6b-H5b)=3.2 Hz,J_(H6b-H6′b)=12.2 Hz); 4.50 (dd, 1H, H6′b, J_(H6′b-H5b)=5.1 Hz,J_(H6′b-H6b)=12.2 Hz); 4.38 (m, 1H, H5a); 4.34 (d, 1H, H2a,J_(H2a-H1a)=3.7 Hz); 4.33 (d, 1H, H3a, J_(H3a-H4a)=3.2 Hz); 4.23 (dd,1H, H4a, J_(H4a-H3a)=3.2 Hz, J_(H4a-H5a)=5.5 Hz); 4.16 (ddd, 1H, H5b,J_(H5b-H4b)=9.7 Hz, J_(H5b-H6b)=3.2 Hz, J_(H5b-H6′b)=5.1 Hz); 4.04 (dd,1H, H6a, J_(H6a-H5a)=6.5 Hz, J_(H6a-H6′a)=8.6 Hz); 3.97 (dd, 1H, H6′a,J_(H6′a-H5a)=5.7 Hz, J_(H6′a-H6a)=8.6 Hz); 1.42, 1.37, 1.25, 1.12 (4s,12H, CH₃).

EXAMPLE 5

This Example Illustrates a Deprotection Reaction which GivesLaminaribiose and which is Carried Out in Two Steps According to theInvention

a) 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→3)-D-glucopyranose

82 g of2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→3)-1,2:5,6-di-O-cyclohexylidene-α-D-glucofuranoseprepared in Example 3 are dissolved in 600 ml of a mixture of equalvolumes of trifluoroacetic acid and water to which 60 ml oftetrahydrofuran are added in order to obtain a good dissolution of theproduct in the reaction medium. After 1 day of reaction, at 40° C., theproduct is precipitated out by diluting the reaction medium with water,filtered, rinsed with water to neutrality, dried and purified bycrystallisation in methanol. 54 g of the desired product were thusobtained.

Yield (%): 80

White solid.

M.Pt. (°C.)=183-186; 197-200 (2 α and β anomers).

TLC: R_(f)=0.6 (dichloromethane/methanol (9/1; v/v)).

¹³C NMR (pyr. d⁵, 101 MHz) δ(ppm): 165.90, 165.87, 165.81, 165.65,165.58, 165.39, 165.37 (C═O); 133.41, 133.22, 133.00, 132.96, 132.93 (Cipso arom.); 130.05-128.28 (C arom.); 101.89, 101.70 (C1b); 98.46(C1aβ); 93.59 (C1aα); 86.48 (C3aβ); 84.69 (C3aα); 77.78 (C5aβ); 75.80(C2aβ); 74.09, 74.06 (C3b); 73.24 (C2aα); 73.04, 72.95, 72.90 (C5aα,C2b); 72.04 (C5b); 70.36, 70.29 (C4b); 69.55, 65.45 (C4a); 63.34, 63.24(C6b); 62.51, 62.36 (C6a).

¹H NMR (pyr. d⁵, 400 MHz) δ(ppm): 8.29-7.09 (3m, 40H, H arom.); 6.58 (t,1H, H3b, J_(H3b-H4b)=J_(H3b-H2b)=9.5 Hz); 6.52 (t, 1H, H3b,J_(H3b-H4b)=J_(H3b-H2b)=9.5 Hz); 6.32 (d, 1H, H1b, J_(H1b-H2b)=8.0 Hz);6.23-6.10 (m, 5H, 2 H4b, H1b, 2 H2b); 5.69 (d, 1H, H1aα,J_(H1aα-H2aα)=3.4 Hz); 5.22 (d, 1H, H1aβ, J_(H1aβ-H2aβ)=7.4 Hz); 4.94(dd, 1H, H6b, J_(H6b-H5b)=2.7 Hz, J_(H6b-H6′b)=12.1 Hz); 4.88-4.69 (m,6H, H6b, H3aα, H6b, H5aα, H6b, H5b); 4.52-4.57 (m, 3H, H6aα, H6aβ, H5b);4.45 (t, 1H, H3aβ, J_(H3aβ-H4aβ)=J_(H3aβ-H2aβ)=9.1 Hz); 4.34-4.26 (m,2H, H6′aα, H6′aβ); 4.20-4.13 (m, 2H, H4aα, H4aβ); 4.10-4.03 (m, 2H,H2aα, H2aβ); 3.97-3.93 (ddd, 1H, H5aβ).

b) Obtaining Laminaribiose

40 g (52.7 mmol; 1 eq) of2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→3)-D-glucopyranose aretreated with 1 l of a solution of sodium methoxide (0.1 eq of sodium)prepared beforehand by dissolving 6 g of sodium in 40 l of anhydrousmethanol. After 4 days of reaction at 35° C., the medium is diluted withwater and then neutralised with acidic resin IR 120, filtered andconcentrated. After lyophilisation, 18 g of laminaribiose are collected.The product can be crystallised in a methanol-water or ethanol-watermixture.

Yield(%): 100

White solid.

M. Pt. (°C.)=199-204 (ethanol-water; Bächli, Percival, J. Chem. Soc.,1952, 1243: 196-206; Takeo, Carbohydr. Res., 1979, 77, 245: 202-204).

M. Pt. (°C.)=124-130 (lyophilised laminaribiose).

[α]_(D) ²⁰=+19.3° (20 min)→+18.7°(24 h; c=1.0; water) (Takeo, Carbohydr.Res., 1979, 77, 245: [α]_(D) ²⁰=+15.5° (10 min)→+18.6° (24 h; c=2.3;water)).

TLC: R_(f)=0.1 (ethyl acetate/isopropanol/methanol/water (10/6/1/1;v/v).

¹³C NMR (DMSO d⁶, 101 MHz) δ(ppm): βanomer: 104.16 (C1b); 96.33 (C1a);88.43 (C3a); α anomer: 104.04 (C1b); 91.78 (C1a); 85.19 (C3a); 76.93,76.87, 76.24, 76.10, 76.07, 73.87, 73.84, 73.50, 71.90, 70.90, 70.18,70.13, 68.66, 68.57, 61.10, 60.92.

¹H NMR (D₂O, 400 MHz) δ(ppm): 5.11 (d, 1H, H1aα, J_(H1aα-H2aα)=3.8 Hz);4.61 (d, 1H, H1b, J_(H1b-H2b)=7.9 Hz); 4.60 (d, 1H, H1b, J_(H1b-H2b)=7.6Hz); 4.55 (d, 1H, H1b, J_(H1aβ-H2aβ)=8.0 Hz); 3.81-3.57 (m, 12H);3.42-3.22 (m, 12H).

EXAMPLE 6

Characterisation of Laminaribiose

In order to characterise the product obtained, the laminaribiosesynthesised was acetylated in the presence of acetic anhydride (12 eq)in pyridine and then recrystallised from ethanol. The β anomer, i.e.1,2, 2′, 3′, 4, 4′, 6, 6′-octa-O-acetyl-β-D-glucopyranose:

was obtained exclusively.

Yield (%): 100

White solid.

M. Pt. (°C.)=165-167 (Takeo, Carbohydr. Res., 1979, 77, 245: 161-162).

[α]_(D) ²⁰=−27° (c=1.0; chloroform) (Takeo, Carbohydr. Res., 1979, 77,245: [α]_(D) ²⁰=−27.6° (c=1.2; chloroform)).

TLC: R_(f)=0.3 (toluene/ethyl acetate (1/1; v/v)).

¹³C NMR (CDCl₃, 101 MHz) δ(ppm): 170.79, 170.57, 170.45, 169.38, 169.37,169.29, 169.20, 168.92 (C═O); 101.02 (C1b); 91.80 (C1a); 78.92 (C3a);72.98, 72.87 (C5a, C3b or vice versa); 71.15 (C2b); 67.99 (C4b); 67.56(C4a); 61.73, 61.71 (C6a, C6b or vice versa); 20.91, 20.88, 20.84,20.74, 20.64, 20.62, 20.52, 20.39 (CH₃).

¹H NMR (CDCl₃, 400 MHz) δ(ppm): 5.61 (d, 1H, H1a, J_(H1a-H2a)=8.4 Hz);5.13 (t, 1H, H3b, J_(H3b-H2b)=J_(H3b-H4b)=9.4 Hz); 5.12 (dd, 1H, H2a,J_(H2a-H1a)=8.4 Hz, J_(H2a-H3a)=9.5 Hz); 5.06 (t, 1H, H4b,J_(H4b-H3b)=J_(H4b-H5b)=9.6 Hz); 5.01 (t, 1H, H4a,J_(H4a-H3a)=J_(H4a-H5a)=9.6 Hz); 4.90 (dd, 1H, H2b, J_(H2b-H1b)=8.1 Hz,J_(H2b-H3b)=9.3 Hz); 4.59 (d, 1H, H1b, J_(H1b-H2b)=8.1 Hz); 4.37 (dd,1H, H6a, J_(H6a-H5a)=4.3 Hz, J_(H6a-H6′a)=12.4 Hz); 4.21 (dd, 1H, H6b,J_(H6b-H5b)=4.6 Hz, J_(H6b-H6′b)=12.4 Hz); 4.13 (dd, 1H, H6′b,J_(H6′b-H5b)=2.2 Hz, J_(H6′b-H6b)=12.4 Hz); 4.05 (dd, 1H, H6′a,J_(H6′a-H5a)=2.2 Hz, J_(H6′a-H6a)=12.4 Hz); 3.93 (t, 1H, H3a,J_(H3a-H2a)=J_(H3a-H4a)=9.4 Hz); 3.78 (ddd, 1H, H5a, J_(H5a-H4a)=10.1Hz, J_(H5a-H6a)=2.2 Hz, J_(H5a-H6′a)=4.6 Hz); 3.68 (ddd, 1H, H5b,J_(H5b-H4b)=9.9 Hz, J_(H5b-H6b)=4.3 Hz, J_(H5b-H6b)=4.3 Hz,J_(H5b-H6′b)=2.3 Hz); 2.12, 2.09, 2.08, 2.03, 2.00, 1.98 (6s, 24H, CH₃).

What is claimed is:
 1. A method of preparing laminaribiose comprising astep of glycosidic coupling between a glycosyl donor and a glycosylacceptor, wherein: the glycosyl donor is in pyranose form and is offormula (II):

 in which: R₁ represents: an alkyl or haloalkyl radical having 1 to 6carbon atoms; an aryl radical which is non-substituted or substitutedwith one or more groups selected from the group consisting of a halogenatom, an alkoxy radical having 1 to 6 carbon atoms and a nitro group; Xrepresents an electrophilic leaving group selected from the groupconsisting of: a group of formula S(O)_(n)R′ in which R′ represents aradical selected from the group consisting of an alkyl radical having 1to 6 carbon atoms, an aryl radical which is non-substituted orsubstituted with an alkoxy group having 1 to 6 carbon atoms, a nitro andacetamide group, and n is an integer equal to 0 or 1 and; atrichloroacetimidate group; the glycosyl acceptor is in furanose formand is of formula (III)

 in which: R₂ and R₃ together form a radical selected from the groupconsisting of a methylidyl, ethylidyl, trichloroethylidyl,isopropylidyl, hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, and 1-phenylbenzylidyl radical; and R₄and R₅ together form a radical selected from the group consisting of amethylidyl, ethylidyl, trichloroethylidyl, isopropylidyl,hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical; orindependently represent a benzyl, acetyl, benzoyl chlorobenzoyl,methoxybenzoyl, nitrobenzoyl, allyl, chlorobenzyl, methoxybenzyl andnitrobenzyl radical; said coupling step is carried out in solution in ananhydrous organic solvent, at a temperature between −80° C. and 40° C.,for a period of 1 minute to 8 hours, in the presence of a suitablepromoter selected from the group consisting of: N-bromosuccinimide orN-iodosuccinimide, combined with a Lewis acid selected from the groupconsisting of ferric chloride, copper ditriflate, tin ditriflate, borontrifluoride dietherate, tin or zirconium tetrachloride, methyl triflate,trimethyl- (or triethyl-) silyl triflate, silver triflate, cadmiumditriflate, cobalt ditriflate, nickel ditriflate, zinc ditriflate,bismuth tritriflate, iron tritriflate, and gallium tritriflate, in thecase in which X represents an S(O)_(n)R′ group as defined above in whichn is equal to 0, a Lewis acid selected from the group consisitng oftriflic anhydride, ferric chloride, copper ditriflate, tin ditriflate,boron trifluoride dietherate, tin or zirconium tetrachloride, methyltriflate, trimethyl- (or triethyl-) silyl triflate, silver triflate,cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zincditriflate, bismuth tritriflate, iron tritriflate, and galliumtritriflate, combined with an amine, in the case in which X representsan S(O)_(n)R′ group as defined above in which n is equal to 1, and aBronsted acid or a Lewis acid selected from the group consisting oftriflic anhydride, ferric chloride, copper ditriflate, tin ditriflate,boron trifluoride dietherate, tin or zirconium tetrachloride, methyltriflate, trimethyl- (or triethyl-) silyl triflate, silver triflate,cadmium ditriflate, cobalt ditriflate, nickel ditriflate, zincditriflate, bismuth tritriflate, iron tritriflate, and galliumtritriflate, in the case in which X represents a trichloroacetimidategroup; and the reaction product thus obtained, neutralised and purified,being subjected to a deprotection treatment to give, after purification,laminaribiose.
 2. The method according to claim 1, wherein: the glycosyldonor is of formula (II) mentioned above in which: R1 represents aradical selected from the group consisting of methyl, chloromethyl,trifluoromethyl, tert-butyl, phenyl, chlorophenyl, methoxyphenyl andnitrophenyl radicals; and X represents a radical selected from the groupconsisting of thiomethyl, thioethyl, thiopropyl, thiophenyl,thionitrophenyl and thiopyridyl radicals.
 3. The method according toclaim 2 wherein: the glycosyl donor is of formula (II) mentioned abovein which: R₁ represents a phenyl radical; and X represents an S(O)_(n)R′radical in which n is equal to 0 and R′ represents an ethyl or phenylradical; the glycosyl acceptor is of formula (III) mentioned above inwhich: R₂, R₃ and R₄, R₅ together form a cyclohexylidyl or isopropylidylradical.
 4. The method according to claim 3, wherein: the promotermentioned above is a mixture of N-iodosuccinimide and of tin ditriflate.5. The method according to claim 3, wherein: the promoter mentionedabove is a mixture of N-iodosuccinimide and of tin ditriflate, inproportions between 1:0.5 and 1:0.005.
 6. The method according to claim1 wherein the deprotection treatment mentioned above comprises: a)cleaving the acetal groups originating from the glycosyl acceptor by atreatment selected from the group consisting of an acidic treatment inan aqueous or hydro-organic medium, and a treatment in the presence ofan acidic resin; b) purifying the product thus obtained; c)transesterifying or hydrolyzing the product obtained in step b); and d)purifying the product thus obtained.
 7. The method according to claim 6,wherein said step a) mentioned above is carried out by allowing theneutralized and purified coupling product to react in a mixture oftrifluoroacetic acid and water, at a temperature between 10 and 70° C.for a period of 1 hour to 10 days, and in that the purification step b)mentioned above is carried out by fractional crystallization.
 8. Themethod according to claim 6, wherein said step a) mentioned above iscarried out by allowing the neutralized and purified coupling product toreact in a mixture of equal volumes of trifluoroacetic acid and water,at a temperature between 10 and 70° C. for a period of 1 hour to 10days, and in that the purification step b) mentioned above is carriedout by fractional crystallization in methanol.
 9. The method accordingto claim 6, wherein the transesterification step mentioned above iscarried out in an alcoholic solvent in the presence of a catalyticamount of an element selected from the group consisting of sodium,sodium methoxide, potassium methoxide, sodium ethoxide and potassiumethoxide for a period of 1 minute to 10 days, and in that thepurification step d) mentioned above comprises: d1) neutralizing theproduct obtained in step c), d2) removing the benzoic ester formed,either by azeotropic evaporation with water, or by extraction with anorganic solvent, d3) concentrating under reduced pressure the residualaqueous phase.
 10. The method according to claim 6, wherein thetransesterification step mentioned above is carried out in an alcoholicsolvent in the presence of a catalytic amount of an element selectedfrom the group consisting of sodium, sodium methoxide, potassiummethoxide, sodium ethoxide and potassium ethoxide for a period of 1minute to 10 days, and in that the purification step d) mentioned abovecomprises: d1) neutralizing the product obtained in step c), d2)removing the benzoic ester formed, either by azeotropic evaporation withwater, or by extraction with an organic solvent, d3) concentrating underreduced pressure the residual aqueous phase, and d4) lyophilizing orcrystallizing the laminaribiose thus obtained in a hydro-alcoholicmixture.
 11. The method according to claim 1, wherein the coupling stepmentioned above is carried out in solution in a solvent selected fromthe group consisting of dichloromethane, 1,2-dichloroethane and toluene,at a temperature between −30° C. and 30° C., for a period of 1 minute to6 hours.
 12. The method according to claim 1, wherein the coupling stepmentioned above is carried out in solution in a solvent selected fromthe group consisting of dichloromethane, 1,2-dichloroethane and toluene,in the presence of molecular sieves, at a temperature between −30° C.and 30° C., for a period of 1 minute to 6 hours.
 13. Laminaribiosesynthesis intermediates, which are selected from the followingcompounds:2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→3)-1,2:5,6-di-O-cyclohexylidene-α-D-glucofuranose

and 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl-(1→3)-D-glucopyranose:


14. A method of preparing laminaribiose comprising a step of glycosidiccoupling between a glycosyl donor and a glycosyl acceptor, wherein: theglycosyl donor is in pyranose form and is of formula (II):

 in which: R₁ represents: an alkyl or haloalkyl radical having 1 to 6carbon atoms; an aryl radical which is non-substituted or substitutedwith one or more groups selected from the group consisting of a halogenatom, an alkoxy radical having 1 to 6 carbon atoms and a nitro group; Xrepresents a trichloroacetimidate group; a the glycosyl acceptor is infuranose form and is of formula (III)

 in which: R₂ and R₃ together form a radical selected from the groupconsisting of a methylidyl, ethylidyl trichloroethylidyl, isopropylidyl,hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, and 1-phenylbenzylidyl radical; and R₄and R₅ together form a radical selected from the group consisting of amethylidyl, ethylidyl, trichloroethylidyl, isopropylidyl,hexafluoroisopropylidyl, cyclopentylidyl, cyclohexylidyl,cycloheptylidyl, butylidyl, 1-tert-butylethylidyl, 1-phenylethylidyl,benzylidyl, methoxybenzylidyl, or 1-phenylbenzylidyl radical, orindependently represent a benzyl, acetyl, benzoyl, chlorobenzoyl,methoxybenzoyl, nitrobenzoyl, allyl, chlorobenzyl, methoxybenzyl andnitrobenzyl radical; said coupling step is carried out in solution in ananhydrous organic solvent, at a temperature between −80° C. and 40° C.,for a period of 1 minute to 8 hours, in the presence of a suitablepromoter selected from the group consisting of a Bronsted acid or aLewis acid selected from the group consisting of triflic anhydride,ferric chloride, copper ditriflate, tin ditriflate, boron trifluoridedietherate, tin or zirconium tetrachloride, methyl triflate, trimethyl-(or triethyl-) silyl triflate, silver triflate, cadmium ditriflate,cobalt ditriflate, nickel ditriflate, zinc ditriflate, bismuthtritriflate, iron tritriflate, and gallium tritriflate, in the case inwhich X represents a trichloroacetimidate group; and the reactionproduct thus obtained, neutralised and purified, being subjected to adeprotection treatment to give, after purification, laminaribiose.